The present invention relates generally to atomic force microscopy and more particularly relates to an atomic force microscope and controller which minimize contact forces between a probe tip and a specimen and is well suited for the study of biological specimens.
In the study of biology, it is desirable to observe biological specimens under very high magnification in a native environment. Such observations allow scientists to monitor, in real time, biological processes at the molecular and sub-molecular level. Such processes include the interaction of proteins with DNA and with each other. Currently, these processes cannot be observed in real time with electron microscopes or x-ray crystallography techniques which are known in the art, as the specimens are not in their native environment when using these apparatuses. Accordingly, scientists have sought alternate methods to observe biological specimens. One such alternative is known as the atomic force microscope.
Atomic force microscopes (AFM), which are generally known in the art, physically probe a specimen to create an image of the specimen""s surface. FIG. 1 illustrates a typical embodiment of an AFM known in the art. The AFM has two primary components, a scanner 10 and a flexible cantilever 12 having a probe tip 14 on a free end. The scanner 10 has a top surface 16 on which a specimen 18 to be imaged is placed. The scanner 10 typically employs three piezoelectric elements 20, 22, 24 to move the specimen 18 in three dimensions, X, Y and Z, relative to the position of the probe tip 14. The probe tip 14 is affixed to the free end of the flexible cantilever 12 and contacts the specimen 18. The AFM includes a laser 26 directed onto the cantilever 12 and a photo detector 28 which is responsive to laser light to measure the deflection of the cantilever 12. As the degree of cantilever deflection is proportional to the contacting force between the probe tip 14 and the specimen 18, such force can accurately be calculated based on the angle of cantilever deflection.
To create an image of a specimen, the scanner 10 directs the specimen 18 in a raster-scan fashion in the X-Y direction while continuously sampling the contour of the specimen 18 in the Z direction. The sampling is generally performed using one of two techniques known in the art, namely contact mode or tapping mode(copyright). (Tapping mode is a registered trademark of Digital Instruments, Inc. of Santa Barbara, Calif.)
In contact mode, the scanner 10 is controlled in the Z direction such that the contacting force between the probe tip 14 and the specimen 18 is substantially constant. As the contour of the specimen changes, the deflection of the cantilever 12 also changes and a servo system driving the scanner 10 adjusts the Z coordinate of the scanner 10 to restore the desired constant force. At each specimen point, the coordinate of the Z axis is indicative of the specimen contour. Because the probe is constantly contacting the surface of the specimen during the X-Y raster scan, significant lateral forces are applied to both the specimen 18 and the probe tip 14. The probe tip 14, which is typically 200-300 angstroms (xc3x85) in diameter is subject to rapid wear and breakage under these forces. Also, when used on soft specimens, such as biological specimens, the probe tip is likely to destroy the surface of the specimen, making accurate and repeatable measurements impossible.
In tapping mode(copyright), the cantilever 12 is driven in an oscillatory fashion at the resonant frequency of the cantilever. This may be achieved by affixing the cantilever to a piezoelectric element 30 and driving the piezoelectric element 30 with a voltage signal at the resonant frequency of the cantilever. To determine the contour of the specimen in tapping mode(copyright), the scanner 10 moves the specimen in the Z direction until a predetermined reduction in oscillation amplitude is detected. The reduction in oscillation amplitude is the result of the probe tip 14 contacting the surface of the specimen 18 during each cycle of oscillation. Because the probe tip 14 only momentarily contacts the specimen 18 during the X-Y raster scan, the lateral force present during contact mode is substantially reduced. However, because the probe tip 14 is moving rapidly on arrival at the specimen surface, the contacting force, while short in duration, is large in magnitude. The force that results from tapping modes tends to be destructive to biological specimens. Thus tapping modes is most useful in sampling hard surfaces, such as those found in integrated circuit manufacturing processes and the like. Also, tapping mode(copyright) is difficult to use when measuring a fluid based specimen. When the cantilever assembly is submerged into a fluid environment, the desired oscillation of the cantilever can be dampened and additional resonances are developed which can adversely affect operation and accuracy. Also, fluid flow induced by the tapping oscillation tends to erode the specimen. Because biological specimens tend to reside in a fluid environment, tapping mode is not well suited for measuring these specimens.
An alternative operating mode to both contact mode and tapping mode(copyright) is described in U.S. Pat. No. 5,229,606 to Elings et al. Elings et al. describe what the inventors refer to as xe2x80x9cjump scanning.xe2x80x9d In jump scanning, the probe is momentarily brought into contact with the surface to be measured. The probe is then lifted away from the surface as the specimen is moved in the X direction and the probe tip is then brought back down into contact to take the next specimen. By jumping over the surface of the specimen, Elings et al. teach a method of increasing scanning speed with reduced risk of probe damage. However, when the probe tip and specimen contact one another, an attractive force tends to hold the probe tip in contact with the specimen. To ensure that the probe tip is able to release, the cantilever 12 must be formed with a sufficient spring constant to overcome this attractive force. Unfortunately, increasing the spring constant of the cantilever 12 increases the magnitude of the contact force between the probe tip 14 and specimen 18 which is required to achieve a measurable cantilever 12 deflection. Such stiff cantilevers, i.e., in the range greater than 0.1 Newtons per meter (N/M), which are required for jump mode, are incompatible with the more sensitive biological specimens which are easily damaged under the application of such forces.
The problem of overcoming the attractive forces between an AFM probe tip 14 and specimen surface was addressed in U.S. Pat. No. 5,515,719 to Lindsay. Lindsay recognized that when soft (low spring constant) cantilevers are used, the adhesive interaction between the specimen 18 and probe tip 14 tends to draw the probe tip in and the probe tip 14 will stick to the surface until enough force is applied to the cantilever base to release the probe tip 14. To address this problem, Lindsay teaches the addition of a magnetic particle attached to the cantilever in combination with a magnetic solenoid located proximate to the cantilever. The solenoid generates a magnetic field which is variable and precisely regulated by a servo circuit. The servo circuit monitors the deflection of the cantilever and continuously adjusts the magnetic field such that the attractive force between the probe tip 14 and specimen 18 is substantially neutralized. In this way, the probe tip 14, as taught by Lindsay, never makes adhesive contact with the specimen. However, in order to operate in a stable fashion, the servo circuit taught by Lindsay must precisely neutralize the attractive force, otherwise instability may result.
The use of a magnetic particle affixed to a flexible cantilever and controlled by a magnetic coil as used by Lindsay was first disclosed in an article by Florin et al., entitled xe2x80x9cAtomic Force Microscope with Magnetic Force Modulationxe2x80x9d, published in the Review of Scientific Instrument, 65(3), March 1994. Florin et al. teach the use of the magnetic control system to drive the cantilever in an oscillating fashion such that the probe tip momentarily contacts a specimen, in a manner similar to tapping mode(copyright).
Current AFM techniques tend to be destructive to biological specimens. Therefore, there remains a need for an improved atomic force microscope adapted for use in a fluid medium for the observation of biological specimens in their native environment.
It is an object of the present invention to provide an atomic force microscope suitable for use with biological specimens in their native environment.
It is another object of the present invention to provide an atomic force microscope which provides a controlled, angstrom by angstrom approach of the probe tip to the specimen.
It is yet another object of the present invention to provide an atomic force microscope which applies minimal vertical force to the specimen being measured.
It is still another object of the present invention to provide an atomic force microscope featuring substantially zero lateral force applied to the specimen during a raster scan.
It is a further object of the present invention to provide an atomic force microscope with a low spring force cantilever which overcomes the problem of probe tip retention resulting from adhesive forces between the probe tip and specimen.
It is still a further object of the present invention to provide an atomic force microscope capable of using a probe tip with a diameter less than 100 angstroms.
It is still another object of the present invention to provide an atomic force microscope which is able to generate repeatable scan to scan measurement results on biological specimens.
It is yet another object of the present invention to provide an atomic force microscope suitable for monitoring biological processes in real time.
It is still another object of the present invention to provide an atomic force microscope which substantially continuously monitors both cantilever displacement relative to a specimen and cantilever deflection.
It is yet another object of the present invention to provide an atomic force microscope which is responsive to changes in cantilever deflection within five microseconds.
It is yet a further object of the present invention to provide an atomic force microscope capable of recording and outputting complete force curves for all pixels in a specimen scan.
In accordance with the present invention, an atomic force microscope (AFM) is provided which addresses the problems known in the prior art for the measurement of biological specimens. The AFM includes a scanner which further includes a specimen surface which is independently moveable along three mutually perpendicular coordinate axes, X, Y and Z. The AFM also includes a compliant cantilever having a probe tip affixed to a free end. The scanner moves the specimen to be imaged relative to the probe tip.
The cantilever has a second end, opposite the free end, which is affixed to a vertically displaceable cantilever base. The cantilever base is responsive to a received approach signal from a controller and moves the cantilever towards or away from the specimen surface along the Z axis. A magnetic particle is affixed to the cantilever proximate the free end. The magnetic particle is responsive to a magnetic field generated by a coil which is energized by the controller. The magnetic particle and coil are arranged such that the magnetic field drives the free end of the cantilever away from the surface of the specimen when tip release is desired.
The atomic force microscope further includes a laser light source which is directed onto the cantilever. A segmented photo sensor receives the laser light reflected from the cantilever and provides a signal which is proportional to cantilever deflection. The signal is provided to the controller to determine the force applied to the probe tip.
The previously described atomic force microscope is preferably operated in accordance with a method of the present invention. The controller increments the X and Y scanner coordinates to effect a raster scan. At each X, Y coordinate, the controller provides an approach signal to move the cantilever base towards the specimen surface. The controller periodically monitors the cantilever deflection during approach and determines when contact has been established between the probe tip and the specimen. When contact is detected, the approach signal reverses, affecting a withdrawal of the cantilever base from the specimen. To disengage the probe tip from the surface of the specimen, the controller initiates a pulse release signal which is applied to the coil. The coil generates a magnetic field which drives the magnetic particle and cantilever tip away from the specimen. The controller monitors cantilever deflection to ensure that the probe tip has released from the specimen. If not, the magnitude of the pulse release signal is increased by the controller and is reapplied to the coil. Alternately, the controller can request intervention by the operator to increase the pulse size for this experiment. While ensuring that the probe tip is clear of the specimen surface, the controller continues to withdraw the cantilever base and increments the scanner to the next X-Y coordinate.
In accordance with another method of the present invention, an AFM is operated in a manner which provides a substantially constant approach distance for successive samples independent of specimen topology. To insure a substantially constant approach distance with varying surface topology, the controller of the present invention compares the last approach value to a predetermined set point. If the approach value is not equal to the set point, the controller drives a Z-axis displaceable element to minimize the error for the next approach. The Z-axis displaceable element can be in the scanner or cantilever base. In this way, the range of cantilever base motion remains substantially constant from scan to scan even with changes in specimen topology.
For better understanding of the present invention, together with other and further objects and advantages, reference is made to the following description, taken in conjunction with the accompanying drawings, and its scope will be pointed out in the appended claims.